Project 3: Pulmonary Vascular-Targeted NO Therapeutic Strategies Pulmonary arterial hypertension (PAH) is a disease of the small pulmonary arteries, characterized by vasoconstriction, vascular proliferation and remodeling. The relaxation of pulmonary vascular smooth muscle cells and their abnormal proliferation is strongly modulated by nitric oxide (NO)-dependent reactions inducing both cGMP-dependent vasodilation and cGMP-independent reactions that inhibit smooth muscle proliferation and inflammatory cell function. Notably, PAH is linked with both decreased NO bioavailability and a lack of responsiveness to NO, a consequence of impaired NO biosynthesis, endothelial nitric oxide synthase (eNOS) uncoupling, dysregulated L-arginine metabolism and increased redox-dependent consumption of NO. We hypothesize that new vascular-targeted, NO-based therapeutic strategies will enhance the treatment of PAH. The research plan will evaluate the mechanisms of action of newly-appreciated signaling mediators in the context of limiting PAH. Specifically, we hypothesize that pulmonary vascular eNOS is negatively regulated by thrombospondin-l. Down-stream of eNOS, NO is then physiologically oxidized to form the potent NO signaling metabolites, nitrite and nitro-fatty acids, which dynamically regulate NO levels, p21 dependent vascular proliferation, phase 2 stress response enzymes, and peroxisome proliferator activating receptor-y signaling. The studies proposed in Project #3 will provide important new mechanistic insight and promising therapeutic strategies for modulating events central to the genesis of PAH. These goals capitalize on recent high impact discoveries related to the formation, metabolism and actions of NOderived species and synergize with central program objectives. Overall, the modulation of eNOS and NO by TSP-CD47 inhibition, nitro-fatty acid supplementation and the therapeutic application of nitrite will be evaluated in a continuum of objectives ranging from basic mechanistic studies to a highly developed translational clinical development program. This development will flow from rodent models of PAH and COPD/PAH, to clinical testing in a Pre-Clinical Core primate model of PAH and in human phase lla catheterization studies in patients with COPD and HIV associated PAH, evaluated in the Clinical Core.